An urgent need exists for the identification and development of novel approaches for delivering protective antigens as more effective vaccines against a variety of agents which might be used as biological weapons, including anthrax, botulism and plague. The long-term objective of the proposed application is to develop a new generation of vaccines against biological agents based on a totally natural, non-reactogenic adjuvant, alpha2-Macroglobulin (alpha2M). Alpha2M has been shown to greatly enhance immunogenicity of a number of antigens. The development of alpha2M adjuvanted vaccines will significantly impact healthcare in both the U.S. and the world, as it will allow a new generation of vaccines, both prophylactic and therapeutic, based on protein subunits, which can be produced inexpensively and are intrinsically safer to use. PA, recognized as the major protective antigen in the current anthrax vaccine (AVA, Anthrax Vaccine Absorbed), will be used as a prototypical subunit candidate for the proposed studies. The specific aims of the proposed studies are to (1) identify the optimal size of Bacillus anthracis PA and the optimal conditions for its covalent incorporation into rabbit alpha2M; (2) determine the ability of various complexes of PA covalently coupled with a2M to generate neutralizing antibodies to anthrax toxin; and (3) determine if the immunogenicity of PA complexed with alpha2M can be enhanced by combination with existing adjuvants. Specifically, full-Iength PA (83 kDa, rPA83) will be expressed in E. coli and purified to homogeneity. Proteolysis of rPA83 will be used to generate "nicked PA" (63 kDa, rPA63), or a carboxy-terminal fragment containing the receptor-binding domain (47 kDa, rPA47). a2M will be purified to homogeneity from rabbit plasma. Studies will determine the efficiency of incorporation of rPA of varying size into rabbit a2M under various conditions. Complexes of rabbit a2M and rPA (alpha2M-rPA) will be then be used to immunize rabbits and will be compared for immunogenicity against rPA alone absorbed onto alum. Sera from immunized rabbits will be evaluated for anti-rPA titers (based on ELISA), immunoglobulin isotypes, and ability to block anthrax toxin mediated macrophage cytotoxicity (neutralizing activity). Alpha2M-rPA complexes, which generate neutralizing titers, will be further evaluated in combination with other adjuvants. These studies will provide a new anthrax vaccine candidate with both improved immunogenicity and decreased reactogenicity.